Imaged capillary isoelectric focusing tandem high-resolution mass spectrometry using nano electrospray ionization (ESI) for protein heterogeneity characterization.

Recombinant monoclonal antibodies (mAbs) have been spurring the rapid growth of commercial biotherapeutics. During production their charge heterogeneity must be assessed as a critical quality attribute to ensure safety, efficacy, and potency. Although imaged capillary isoelectric focusing (icIEF) is a powerful tool for this process, it could be improved further with tandem high-resolution mass spectrometry (HRMS). In this work, a nano-electrospray ionization (nano-ESI) apparatus was constructed to directly couple icIEF to HRMS. The system was evaluated with the standard NISTmAb, as well as more complex mAb, bi-specific antibody, and fusion protein samples. NISTmAb concentrations as low as 0.25 mg/ml demonstrated excellent sensitivity. There were good repeatabilities at 1 mg/ml with 7.58% and 8.01% RSDs for intention time and MS intensity, respectively, and the HRMS signal showed a strong linearity (R = 0.9983) across different concentrations. Meanwhile, the fingerprinting of the complex samples illustrated the versatility and potential of icIEF-HRMS. icIEF-HRMS developed can provide a comprehensive understanding of the underlying structural modifications that impact protein charge heterogeneity. Compared to the traditional ESI, nano-ESI can significantly improve sensitivity while maintaining a reasonable repeatability and throughput. Furthermore, the interface is much easier to connect, and is compatible with many commercial HRMS instruments.

Fractionation and online mass spectrometry based on imaged capillary isoelectric focusing (icIEF) for characterizing charge heterogeneity of therapeutic antibody.

Imaged capillary isoelectric focusing (icIEF) technology has been proved to be robust for the characterization of protein charge heterogeneity due to its high-resolution pI discrimination and high-throughput. Although high performance liquid chromatography (HPLC) tandem mass spectrometry (MS) and offline fraction collection technologies including isoelectric focusing (IEF), ion exchange chromatography (IEX) and free flow electrophoresis (FFE) have been widely utilized for protein charge variant characterization, there are a few applications of MS coupling with icIEF as a front-separation technique and related fractionation technologies for protein charge heterogeneity. However, the application of icIEF-MS has been much less frequent due to difficulties in MS interface, compatible ampholyte and coated capillary cartridge designation, ultimately impeding the breadth of icIEF applications in protein charge heterogeneity. In this study, a therapeutic monoclonal antibody (mAb-M-AT) was used for its charge variant characterization on an integrated icIEF platform with functions including analytical profiling, MS online coupling and fraction collection for charge heterogeneities. The main protein component and its four charge variants were identified using direct icIEF-MS coupling. Additionally, the two major acidic and basic charge variants were collected using preparative fractionation after the protein focused in the separation capillary. The identity of the fractions was confirmed by LC-MS at intact protein level and the results were consistent with those using icIEF-MS online coupling. The multiple operation modes of the icIEF platform described above can be rapidly and flexibly switched just by changing customized capillary separation cartridges without drastically altering instrument configuration. The whole workflow of icIEF-based profiling, fractionation and MS online coupling for protein heterogeneity is straightforward, reliable, and accurate, thus providing comprehensive solutions for in-depth protein heterogeneity characterization.

Imaged capillary isoelectric focusing (icIEF) tandem high resolution mass spectrometry for charged heterogeneity of protein drugs in biopharmaceutical discovery.

Since the first commercial imaged capillary isoelectric focusing (icIEF) instrument was developed twenty years ago, the technology has become the gold standard of quality and manufacturing process control in the biopharmaceutical industry. This is owing to its high-resolution and high-throughput characterization of protein charge heterogeneity. In addition to a charge variant profiling, mass spectrometry (MS) analyses are also desirable to obtain an in-tact molecular weight (MW) and further identification of these charged species. While offline fractionation technologies including isoelectric focusing (IEF) and free flow electrophoresis (FFE) followed by liquid chromatography (LC)-mass spectrometry (MS) coupling have been employed for this purpose, there have been much fewer reported applications of icIEF-based MS connection and fraction collection. Factors that have impeded the development of these icIEF applications include difficulties with a direct connection to the MS interface as well as high background signal of carrier ampholytes and incompatible coated capillary cartridges. In this work, we developed a robust and flexible icIEF-MS platform which overcomes these challenges to achieve both the rapid icIEF separation and high-resolution MS (HRMS) identification of protein charged variants simultaneously. We demonstrate how this methodology proves highly-sensitive and highly reliable for the characterization of commercial monoclonal antibodies (mAbs) and antibody-drug-conjugates (ADCs). The whole workflow of icIEF-MS for protein heterogeneity is straight forward and accurate and can be performed within 45 min. Furthermore, the developed icIEF-MS configuration can flexibly switch to icIEF-based fraction collection model allowing the user to perform additional in-depth characterization such as peptide mapping by high performance liquid chromatography (HPLC) tandem mass spectrometry (LC-MS/MS).